In the last 10 to 15 years, two methods have prevailed in mass spectrometric analysis for the soft ionization of biological macromolecules: ionization by matrix-assisted laser desorption (MALDI), and electrospray ionization (ESI). The biological macromolecules analyzed will be termed analyte molecules below. With the MALDI method, the analyte molecules are generally prepared on the surface of a sample support in a solid matrix, whereas with the ESI method they are dissolved in a liquid. Both methods have a considerable influence on the mass spectrometric analysis of biological macromolecules in genomics, proteomics and metabolomics; their inventors were awarded the Nobel prize for chemistry in 2002.
In a prepared MALDI sample, there are 103 to 105 times more matrix molecules than analyte molecules, and they form a polycrystalline, solid matrix in which the analyte molecules are integrated, either scattered in the interior of the crystals or at their grain boundaries. The prepared MALDI sample is irradiated with a short laser pulse, which is strongly absorbed by the matrix molecules. By the pulsed irradiation, the solid matrix is explosively transferred into the gaseous phase of a vaporization cloud (desorption). The analyte molecules are usually ionized by being protonated or deprotonated in reactions with matrix molecules or matrix ions, the analyte ions being predominantly singly charged after leaving the vaporization cloud. The degree of ionization of the analyte molecules is only around 10−4. The MALDI method is termed soft ionization because an analyte molecule is transferred in isolation into the gaseous phase and ionized, without undergoing dissociation.
Despite the linear absorption by the matrix, matrix-assisted laser desorption/ionization is a nonlinear process, which only starts above an intensity threshold of around 106 watts per square centimeter using laser pulses with duration of a few nanoseconds. For soft ionization, the maximum intensity lies at an upper limit of approximately 107 watts per square centimeter. With a typical duration of around ten nanoseconds, the stated intensity limits produce a fluence of between 10 and 100 millijoules per square centimeter.
The MALDI process is complex and affected by numerous factors, some of which are interdependent. Since the MALDI method was first published in 1988, many chemical and physical parameters have been investigated and varied. The chemical parameters are, for example, the type of matrix substances themselves, the concentration ratio between matrix and analyte molecules and the preparation conditions. As far as the physical parameters are concerned, until now the temporal duration of the laser pulses, the intensity in the laser focus and the wavelength of the pulsed laser beam have mainly been considered. In spite of intensive research, the processes in the matrix and in the vaporization cloud which lead to the ionization of the analyte molecules are still not completely understood (K. Dreisewerd, Chem. Rev. 103 (2003), 395-425: “The Desorption Process in MALDI”).
Nowadays, pulsed laser systems in the ultraviolet spectral range (UV) are used in the vast majority of MALDI mass spectrometers. In principle, various laser types and wavelengths A are available in the UV: nitrogen lasers (λ=337 nm), excimer lasers (λ=193 nm, 248 nm, 308 nm) and Nd:YAG lasers (λ=266 nm, 355 nm). Commercially, however, only the nitrogen laser with a wavelength of 337 nanometers and the Nd:YAG laser at 355 nanometers are of interest, the nitrogen laser being far and away the one most frequently used. The duration of the laser pulses used in the UV MALDI mass spectrometers is typically between 1 and 20 nanoseconds.
Depending on the chemical substance classes, for example proteins or nucleic acids, over one hundred different chemical matrix substances are known for the analyte molecules, such as sinapic acid, DHB (2,5-dihydroxy benzoic acid), CHCA (α-cyano-4-hydroxy cinnamic acid) or HPA (3-hydroxypicolinic acid). All these matrix substances exhibit strong absorption in the wavelength range between 330 and 360 nanometers. Furthermore, a MALDI sample can be prepared in a number of different ways, depending on the application, for example with the “dried droplet” preparation method or thin layer preparation.
Nitrogen lasers are most widely used in MALDI mass spectrometry at present. The laser medium is gaseous nitrogen, which is excited by means of an electrical discharge between two electrodes. The most intensive laser line at 337 nanometers exhibits such a high amplification that a single laser pulse can reduce the population inversion of the energy states even if it sweeps the electrodes only once. Even when cavity mirrors are used, numerous transverse beam modes are excited and superimpose themselves in the beam profile of the laser beam. From the field of optics we are familiar with the fact that a laser beam of this type cannot be brought to a diffraction-limited focus. At a wavelength of 337 nanometers, the laser focus therefore has a minimum diameter of only three micrometers. However, the diameter of the irradiated area on the MALDI sample is typically between 20 and 200 micrometers. The beam profile of the nitrogen laser has an almost rectangular flat top at the electrodes, the width and the height of the beam profile being determined by the width of the discharge electrodes and their separation respectively. In principle, an electrical gas discharge is not equal at all points between the electrodes, resulting in a spatially inhomogeneous amplification. The short time the laser is in action means that this inhomogeneity is not evened out but is instead transferred to the beam profile of the nitrogen laser. Consequently, although the nitrogen laser has a flat-top beam profile when integrated over many laser shots, the profile is spatially modulated in the single shot and exhibits intensity maxima and minima.
A disadvantage of the nitrogen laser is that the repetition rate of the laser pulses is limited to around 100 hertz unless provision is made for a rapid gas exchange. In MALDI mass spectrometers the nitrogen lasers are therefore usually operated at a repetition rate of 50 Hertz at the most. More serious than the limited repetition rate, however, is the short life time. For commercially available nitrogen lasers the life time is around 107 laser pulses before maintenance is necessary. The life time of the nitrogen laser is presumably limited by the fact that the electrical gas discharge causes wear to the electrodes and the laser medium. With a laser pulse repetition rate of 50 Hertz and a daily operating time of only one hour this means the life time is just two months, very low by industrial standards. Furthermore, a pulsed gas discharge is normally difficult to reproduce, so that the intensity distribution in the beam profile and the energy fluctuate from laser pulse to laser pulse.
The great advantage of the nitrogen laser in MALDI mass spectrometry is that a large number of suitable matrix substances are available for this type of laser, for example sinapic acid, DHB or CHCA. The matrix substances and the preparation specifications which have been compiled for them are adapted for different applications and classes of analyte molecules. The wavelength of the nitrogen laser seems to lie in the best possible region for soft ionization, a fact which is borne out not least by the prevalence of the nitrogen laser.
The quality of a MALDI mass spectrum generally increases with the absorption of the MALDI matrix, although differences will level out, if the absorption exceeds a certain value (Dreise-werd: “The Desorption Process in MALDI”, Chem Rev, 103, 2003). Above a wavelength of 380 nanometers there is a serious decrease of absorption and thus a loss of performance in the MALDI process for standard matrix substances, such as CHCA or DHB. A lower limit is imposed by the undesirable excitation of electronic states in the aromatic rings of the matrix and analyte molecules. On the basis of measured absorption spectra of the matrix substances, it can be estimated that the absorption remains constant if the wavelength of the laser pulses deviates less than five nanometers from the wavelength of the nitrogen laser at 337 nanometers. With a wavelength difference of less than two nanometers there are no wavelength-specific differences for the MALDI process whatsoever.
In addition to the nitrogen laser, the Nd:YAG laser is also used in MALDI mass spectrometers. The Nd:YAG is a solid-state laser whose laser medium is a YAG crystal (yttrium aluminum garnet:Y3Al5O12) doped with neodymium ions. The strongest and most frequently excited laser line lies at a wavelength of 1064 nanometers. This laser frequency can be doubled, tripled or quadrupled by non-linear optical processes, so that in addition to the fundamental wavelength of 1064 nanometers “new” wavelengths at 532 nanometers, 355 nanometers and 266 nanometers arise. The tripled fundamental frequency at a wavelength of 355 nanometers is the one which is almost always used in MALDI mass spectrometers. Solid-state lasers often have a spatial beam profile consisting of one transverse fundamental mode or a small number of transverse beam modes. If this type of laser beam is focused or imaged onto the sample, then there is a Gaussian or almost Gaussian intensity distribution with a single maximum (intensity peak) on the sample. The half-width of the intensity peak is the maximum separation between two points (on the sample) at which the intensity of the maximum has fallen to half the value. In the UV, the half-width of an intensity peak can be less than one micrometer.
The great advantages of the Nd:YAG laser compared with the nitrogen laser are the high repetition rate of the laser pulses, the low energy fluctuations between individual laser pulses and the long life time. The repetition rate can be over 100 kHz. If an Nd:YAG crystal is excited by a diode laser, the life time of a pulsed Nd:YAG laser is around 109 laser pulses. This makes the life time of the Nd:YAG laser a hundred times longer than that of a typical nitrogen laser.
Previous experience has shown that the disadvantage of the Nd:YAG laser is that it is less efficient than the nitrogen laser in most MALDI applications. One possible reason is that the wavelength of 355 nanometers which is used differs by 18 nanometers from the wavelength of the nitrogen laser. Changing the laser systems in the MALDI mass spectrometer from a nitrogen laser to a Nd:YAG laser is usually very time-consuming for the user since the operating specifications which have been compiled and optimized for the nitrogen laser have to be re-evaluated.
In U.S. Pat. No. 6,953,928 B2 Vestal et al. disclose a MALDI mass spectrometer utilizing a Nd:YAG laser. They state once only in the description that the Nd:YAG laser has a wavelength of 335 nanometers. It is evident that the wavelength of 335 nanometers is a typographical error, since the used MALDI mass spectrometer is a commercially available 4700 Proteomics Analyzer by Applied Biosystems® working at a standard wavelength of 355 nanometers as can be seen in the manual. A Nd:YAG laser emitting at a wavelength of 335 nanometers is not available at present.
For the mass spectrometric analysis of the analyte ions generated in the MALDI process, both conventional sector field mass spectrometers and quadrupole mass spectrometers as well as quadrupole ion trap mass spectrometers and ion cyclotron resonance mass spectrometers are possible, in principle. Particularly suitable, however, are time-of-flight mass spectrometers with axial injection which require a pulsed current of ions to measure the time-of-flight (TOF). In this case, the starting time for the time of flight measurement is dictated by the ionizing laser pulse. The MALDI process was originally developed for use in a vacuum. In more recent developments, matrix-assisted laser desorption/ionization is also used at atmospheric pressure (AP MALDI ). Here, the ions are generated with a repetition rate of up to 2 kilohertz and can be fed with the help of an ion guide to a time-of-flight mass spectrometer with orthogonal injection (OTOF—“orthogonal time-of-flight”), a quadrupole ion trap mass spectrometer or an ion cyclotron resonance mass spectrometer. In an OTOF mass spectrometer, the ions generated in the MALDI process can be fragmented and stored before measurement of the time-of-flight with electronic pulsed ejection is commenced.
There are imaging mass spectrometric analytical methods (IMS), in which the MALDI process is used to generate the ions. With IMS, a thin section of tissue obtained, for example, from a human organ using a microtome, is prepared with a matrix substance and analyzed spatially resolved using a mass spectrometer. The spatial resolution of the mass spectrometric analysis can be achieved either by scanning individual spots of the tissue section or by stigmatic imaging of the ions generated. With the scanning method the pulsed laser beam is focused onto a small diameter on the sample, and a mass spectrum is measured for each individual pixel. The spatial distributions (in one or two dimensions) of individual proteins are produced from the plurality of individual spatially resolved mass spectra. With stigmatic imaging in a TOF mass spectrometer, an area of up to 200 by 200 micrometers is irradiated homogeneously with a laser pulse. The ions generated in this way are imaged ion-optically pixel by pixel onto a spatially resolving detector. Until now it has only been possible to scan the spatial distribution of one ion mass with a single laser pulse because no spatially resolving ion detectors are available that are fast enough for. The measured ion mass can, however, be varied from laser pulse to laser pulse.